The scope of the proposed research is to study mammalian lysosomal neuraminidase (neur), an essential hydrolase that belongs to the superfamily of sialidases found widely spread in nature. Information on the mammalian neuraminidases, including a cytosolic, a lysosomal and a plasma membrane form, has been limited by their apparent low abundance, instability and membrane-bound character. Only in recent years important clues have emerged as to the physiological function(s) of these enzymes. Lysosomal neur initiates the hydrolysis of sialo- glyconjugates by removing their terminal sialic acid residues. The enzyme is unique among sialidases in that it requires association with the protective protein/cathepsin A (PPCA) for intracellular routing and lysosomal activation, and it is associated with neurodegenerative diseases of metabolism. Sialidosis is caused by structural lesions at the neur locus, and galactosialidosis (GS), a combined deficiency of neur and beta- galactosidase (beta-gal) is caused by the absence of PPCA. The two diseases have common clinical and biochemical features that can be assigned to the loss of neur function. Our overall goal is to gain a broader understanding of lysosomal neur function(s), and to apply this knowledge to the study of the pathophysiology of sialidosis and GS which, in turn, could help in designing appropriate therapy. We plan to investigate the structure- function relationships between neur and PPCA, making use of the 3D structure of the PPCA precursor for targeted mutagenesis of potential contact sites between the two proteins, and of natural neur mutation(s) identified in patients with sialidosis. With this approach we will identify residues/domains crucial for neur/PPCA interaction, intracellular transport and activation. We will also biochemically characterize a novel neuraminidase isoform with the idea of linking specific biochemical properties with function. The characterization of neur knockout mice will be instrumental to determine the general penetrance of the disease that results from neur deficiency, and to compare it with the GS mouse. These in vitro and in vivo studies will be coupled to the determination of neur 3D structure using the baculovirus expression system to produce neur protein in large quantities. Finally, provided that we will be successful in generating stable complex of neur and PPCA we will initiate crystallization of the complex of the two enzymes. The PI's laboratory is in the unique position to develop this line of investigation as it has established genetic and biochemical systems for the proposed studies, and can rely on the expertise of an outstanding structural biologist for the crystallography part of the project.